Jianyong Li

Involvement of l-tyrosine and phenol oxidase in the tanning of Aedes aegypti eggs

Abstract l -Tyrosine was found to be the initial precursor involved in the tanning of the chorion of Aedes aegypti eggs. This amino acid became detectable at about 24 h post-blood feeding and significant amounts of this compound accumulated in the ovaries during subsequent egg development. However, no o-diphenolic compounds were synthesized during the development of eggs within the mosquito ovaries. Oviposition of eggs to the outside environment initiated eggshell tanning due to activation of phenol oxidase, which then catalyzed the production of l -DOPA from l -tyrosine and further oxidized l -DOPA and dopamine to their respectivi o-quinones. Hardening and blackening of the chorion was completed in about 4 h. DOPA decarboxylase (DDC) also seemed to be involved in chorion tanning because a high percentage of l -DOPA, produced by the action of phenol oxidase following its activation, was decarboxylated to dopamine in in vitro assays. In addition, the application of the DDC inhibitor, m-hydroxybenzylhydrazine, resulted in an increased time required for the complete tanning of the chorion.

Biochemical pathway of melanotic encapsulation of Brugia malayi in the mosquito, Armigeres subalbatus

The mosquito, Armigeres subalbatus, is naturally resistant to the filarial worm, Brugia malayi, and microfilariae (mf) penetrating the midgut are killed by melanotic encapsulation reactions in the hemocoel within 48 h following ingestion. This vector-parasite system was used to assess changes in hemolymph tyrosine, tyrosine derivatives, and catecholamine-metabolizing enzyme activities using high pressure liquid chromatography with electrochemical detection (HPLC-ED) during melanotic encapsulation reactions against mf. Tyrosine and dopa were detected in the hemolymph of both control and immune-activated (mf-exposed) mosquitoes, but not dopamine or N-acetyl dopamine (NADA). Tyrosine was significantly increased in immune-activated mosquitoes at 6 and 12 h post blood feeding, but was depleted following intrathoracic inoculation of mf in the absence of a blood meal. Dopa also was elevated in immune-activated mosquitoes at 6, 12, and 24 h post-exposure to mf. There were significant increases in phenol oxidase (PO) and dopa decarboxylase (DDC) activities in immune-activated mosquitoes as compared to controls, and these elevated activities were correlated with changes in tyrosine and dopa levels in the hemolymph. No significant differences in N-acetyl transferase (NAT) and dopachrome conversion enzyme (DCE) activities between control and immune-activated mosquitoes were observed. The possible roles these molecules play in melanotic encapsulation reactions of A. subalbatus against mf are discussed.

Biogenic monoamines in the freshwater snail, Biomphalaria glabrata: influence of infection by the human blood fluke, Schistosoma mansoni.

The biogenic monoamines, serotonin (5-HT), dopamine (DA) and L-dopa were measured using high performance liquid chromatography with electrochemical detection (HPLC-ED) in the extracts of the central nervous system (CNS) and plasma of uninfected freshwater snails, Biomphalaria glabrata, and in snails at 7, 14, 21 and 28 days postexposure (PE) to the miracidia of the human blood fluke, Schistosoma mansoni. Relative to age-matched uninfected snails, a general depression of biogenic amine levels was observed in the plasma (cell-free haemolymph) and the CNS of infected snails, especially during the latter phase of the prepatency period. Significant decreases were first observed in the CNS of infected snails beginning at Day 14 PE for DA and 5-HT and Day 21 PE for L-dopa. Parasite-exposed snails also exhibited an early and persistent suppression of plasma 5-HT concentrations, starting at 7 days PE and continuing throughout the infection test period. In order to determine the effect of 5-HT on reproduction and, thereby, establish a possible relationship between the observed parasite-induced reduction in 5-HT levels and parasitic castration, the effect of exogenous 5-HT on individual infected and uninfected B. glabrata was investigated. Repeated treatment with 10 microM 5-HT promoted both ovulation and oviposition in B. glabrata. Snails treated with 5-HT consistently layed more eggs than did sham-treated controls. Infected snails that were treated with 5-HT exhibited similar egg-laying rates as those of both serotonin-treated and untreated, uninfected snail groups, thus reversing the castrating effects of larval infection. These findings suggest that 5-HT acts as a stimulant for egg production in B. glabrata, and that parasitic castration may be due, at least in part, to larval-induced suppression of 5-HT in the snails CNS and plasma during the course of infection with S. mansoni.

Effect of pH on the oxidation pathway of dopamine and dopa

The formation of 6-hydroxylated o-diphenolic compounds and other intermediates during the oxidation of dopamine, dopa and 3[3,4-dihydroxyphenyl]propionic acid by NaIO4 or mushroom tyrosinase was studied using high pressure liquid chromatography with electrochemical detection. The results suggest the following: (1) acidic conditions are necessary to achieve accumulation of both dopaquinone and dopaminequinone during dopa and dopamine oxidation by NaIO4 and also seem essential in the o-quinone hydroxylation pathway; (2) accumulation of o-quinones probably results in the formation of semiquinone owing to the interaction between o-quinone species and o-diphenolic precursors; (3) quinone methide is probably the reactive intermediate involved in the formation of 6-hydroxylated o-diphenolic compounds; (4) rapid hydroxylation of dopaquinone is due to the effect of the side chain carboxy group by enhancing the release of the β proton, thereby facilitating the tautomerization of dopaquinone to quinone methide; (5) neutral buffer promotes both the cyclization pathway of dopaquinone and dopaminequinone to form their respective aminochromes and the structural rearrangement of these aminochromes to 5,6-dihydroxyindole.

Dopachrome conversion activity in Aedes aegypti: Significance during melanotic encapsulation of parasites and cuticular tanning

Phenol oxidase (PO) and dopachrome conversion enzyme (DCE) were partially purified from Aedes aegypti larvae by ammonium sulfate fractionation. PO from A. aegypti functions in the hydroxylation of monophenols (e.g., tyrosine and tyramine) to their related o-diphenols, and the oxidation of o-diphenols (e.g., L-dopa, dopamine, N-acetyldopamine) to their respective o-quinones. Partially purified DCE showed high specificity toward dopachrome generated from dopa with the L-configuration. The combined effects of PO and DCE significantly accelerated melanization pathways when L-dopa was used as substrate. Significant DCE activity also was detected in hemolymph samples from adult, female A. aegypti, and undoubtedly plays a role in melanotic encapsulation reactions.

Phenol oxidase activity in hemolymph compartments of Aedes aegypti during melanotic encapsulation reactions against microfilariae

Monophenol oxidase (MPO) and diphenol oxidase (DPO) activity was assessed in hemocytes, cell-free plasma and complete hemolymph collected from Aedes aegypti Liverpool strain, intrathoracically inoculated with saline alone, immune activated by the inoculation of Dirofilaria immitis microfilariae (mff), and uninoculated. Enzyme activities between groups were compared using a radiometric hydroxylation assay (MPO) and a high pressure liquid chromatography with electrochemical detection assay (DPO). There were no significant differences in enzyme activity in hemocytes, cell-free plasma, and complete hemolymph between uninoculated and saline-inoculated controls. Both MPO and DPO activity of mosquito hemocytes and complete hemolymph from immune-activated mosquitoes were significantly increased at 12 and 24 h postinoculation as compared with the enzyme activity from saline-inoculated mosquitoes, but no significant increase in enzyme activity was detected in cell-free plasma from immune-activated mosquitoes. Increases of MPO and DPO activity in hemocytes and hemolymph following immune activation were proportional, thereby suggesting that a single enzyme might react with both monophenols and o-diphenols within the hemolymph of A. aegypti. Results also suggest that augmented phenol oxidase activity associated with melanotic encapsulation reactions is associated primarily with hemocytes.

PHENOLOXIDASE ACTIVITY IN THE REPRODUCTIVE SYSTEM AND EGG MASSES OF THE PULMONATE GASTROPOD, BIOMPHALARIA GLABRATA

Phenoloxidase (PO) activity in the albumen gland (AG) and egg masses (EM) of Biomphalaria glabrata was assessed using high-performance liquid chromatography combined with electrochemical detection and colorimetric techniques. Both AG and EM extracts catalyzed the hydroxylation of L-tyrosine (monophenol oxidase activity, MPO) and oxidation of L-dopa (diphenol oxidase activity, DPO). However, no PO activity was found in the ovotestis. Both MPO and DPO activities in AG and EM were significantly inhibited by 1-phenyl-2-thiourea and inactivated by boiling. Approximately 35% of MPO and 44% of DPO activities were detected in the soluble fraction of homogenized EM, in contrast to that of homogenized AG, which contained about 5% and 12%, respectively, of MPO and DPO activities. N-acetyl-dopamine, a diphenolic compound, enhanced the hydroxylation of tyrosine by the PO. The presence of both MPO and DPO activities also was confirmed by the accelerated accumulation of dopachrome during incubation of EM extracts with L-tyrosine in the absence of ascorbate. Temperature and pH optima for this enzyme were 30 degrees C and 7.5, respectively. The potential roles of PO in egg formation in B glabrata are discussed.

Electrochemical determination of diphenol oxidase activity using high-pressure liquid chromatography☆

A quantitative assay for the diphenol oxidase activity of tyrosinase (EC 1.14.18.1) using high-pressure liquid chromatography with electrochemical detection is described. The assay is based on the observation (M. Sugumaran, 1986, Biochemistry 25, 4489-4492) that tyrosinase catalyzes the oxidative decarboxylation of 3,4-dihydroxymandelic acid to 3,4-dihydroxybenzaldehyde. The substrate and product were readily separated on a reverse-phase column equilibrated with 0.1 M citrate buffer, pH 3.2, containing 0.5 mM Na2 EDTA, and 5% (v/v) acetonitrile. The reaction of DHMA with mushroom tyrosinase was linear with time and proportional to the amount of enzyme present. The specific activity of mushroom tyrosinase using the method was about fourfold greater than that obtained using a spectrophotometric assay for diphenol oxidase following dopachrome formation from L-3,4-dihydroxyphenylalanine. The applicability of the high-pressure liquid chromatographic assay to determination of diphenol oxidase activity in small biological sample sizes was demonstrated by using microgram quantities of crude, cell-free hemolymph from Aedes aegypti mosquitoes.

Identification of Products and Intermediates During L-Dopa Oxidation to Dopachrome Using High Pressure Liquid Chromatography with Electrochemical Detection

Abstract Products and intermediates produced during L-dopa oxidation by sodium periodate and mushroom tyrosinase were studied using high pressure liquid chromatography with electrochemical detection (HPLC-ED). The oxidation products and intermediates, including dopaquinone, leukodopachrome, 2, 4, 5-trihydroxyphenylalanine and its quinone species, were readily separated on a reverse-phase column with 15 mM citrate buffer, pH 3.0, containing 35 mM NaCl, 0.25 mM octyl sulfate and 0.7 mM Na2EDTA and detected at either a reductive (-100 mV) or an oxidative potential (750 mV) of the working electrode. Using these techniques, the effect of base and acid on the chemical reaction of dopaquinone to dopachrome was demonstrated.

Involvement of lactones in the formation of 6-hydroxydopa and 6-hydroxyhydrocaffeic acid during oxidation of dopa and hydrocaffeic acid

Detailed oxidation/reduction pathways that lead to the formation of 2,4,5-trihydroxyphenolic compounds during the oxidation of dopa, hydrocaffeic acid and N-acetyldopa are described. Data suggest that (i) oxidation of dopa, hydrocaffeic acid and N-acetyldopa results in the formation of their corresponding o-quinones, (ii) the o-quinones undergo intramolecular nucleophilic addition through the side chain carboxy group to form lactones under weak acid conditions, (iii) oxidation of the lactones produces their corresponding lactone-o-quinones, (iv) the lactone-o-quinones, once formed, undergo rapid hydrolysis that leads to the formation of 2-hydroxy-1,4-benzoquinones (p-quinones) as relatively stable products and (v) reduction of the p-quinones results in the formation of their corresponding 6-hydroxydopa, 6-hydroxyhydrocaffeic acid and N-acetyl-6-hydroxydopa. Data suggest that 6-hydroxydopa also can be produced by direct hydrolysis of dopa-lactone.